Synthetic Pseudomonas aeruginosa pilin peptide vaccine

ABSTRACT

Treatment and diagnosis of P. aeruginosa infection or colonization is achieved in accordance with this invention by the discovery of a polypeptide which is smaller than the naturally occurring P. aeruginosa pillin protein. The pure polypeptide comprises at least one amino acid residue sequence containing about twelve amino acid residues and up to about twenty amino acid residues that define a sequence capable of immunologically mimicking an antigenic determinant cite of P. aeruginosa pilin. This amino acid residue sequence can repeat as a unit one or more times in the same polypeptide molecule. More than one of such repeating units and more than one repeating unit of the same type can be present in a single polypeptide molecule. The polypeptides act an antigens or immunogens and antibodies may be raised to the immunogens and a vaccine prepared suitable for the prevention of P. aeruginosa infection.

This is a continuation of application Ser. No. 07/927,797 filed on Aug.10, 1992, now U.S. Pat. No. 5,445,818, which is a continuation ofapplication Ser. No. 07/344,565 filed Apr. 28, 1989 abandoned.

FIELD OF THE INVENTION

This invention relates to antigens, immunogens and to vaccines utilizingsuch immunogens. More particularly, this invention relates topolypeptide antigens or immunegens, antibodies raised by suchimmunogens, and a vaccine suitable for the prevention of P. aeruginosainfection or colonization.

BACKGROUND OF THE INVENTION

During the past two decades, Pseudomonas aeruginosa has been recognizedas a pathogen which causes between 10% and 20% of infections in mosthospitals. Pseudomonas infection is especially prevalent among patientswith burn wounds, cystic fibrosis, acute leukemia, organ transplants,and intravenous-drug addiction. P. aeruginosa is a common nosocomialcontaminant, and epidemics have been traced to many items in thehospital environment. Patients who are hospitalized for extended periodsare frequently affected by this organism and are at increased risk ofdeveloping infection. The most serious infections includemalignant-external otitis, endophthalmitis, endoconditis, meningitis,pneumonia, and septicemia. The likelihood of recovery from Pseudomonasinfection is related to the severity of the patient's underlying diseaseprocess. The reported mortality for P. aeruginosa pneumonia is as highas 50-80%. Even with the development of newer antibiotics, resistanceremains a problem necessitating combined antibiotic treatment for severeP. aeruginosa infections.

Alternative therapy for the management of severe P. aeruginosainfections have been evaluated for many years. Immunotherapy has beenthe alternative most extensively explored. In this area, attention hasfocussed on the virulence factors. As with most bacterial pathogens,virulence of Pseudomonas aeruginosa is multifactorial and is the productof many interacting variables, involving both the bacterium and thehost.

Evidence suggests that the initial event in infection is the adherenceof microorganisms to epithelial cells of mucosal surfaces [E. H.Blackey, J. Infect. Dis., 143: 325-345 (1981)]. Organisms that areunable to adhere to mucosal surfaces fail to colonize because they areremoved by the secretions that bathe the mucosal surfaces. The adherenceprocess is dependent upon the specific recognition between bacteria andepithelial cells. For a number of gram-negative bacteria, including P.aeruginosa, attention has been directed to surface appendages asmediations of adherence. These surface appendages are termed `adhesins`,and the distribution of specific receptors for adhesins determines manyof the tissue tropisms noted for bacteria. In the case of P. aeruginosa,polar pili present on the surface of the organism have been shown tomediate adherence to buccal epithelial cells. The evidence for this isas follows: (1) nonpiliated strains do not adhere to epithelial cells;(2) protease treatment of P. aeruginosa drastically reduces the abilityof these organisms to adhere to epithelial cells; (3) preincubation ofepithelial cells with purified pili significantly decreases theadherence of intact organisms, and (4) antibody to purified piliprevents the adherence of organisms to buccal epithelial cells.

Although P. aeruginosa pili are antigenically heterogeneous in differentclinical isolates, there is evidence that a portion of the pilus isconserved [see Paranchych et al., Antibiotics Chemother. 36: 49-57(1985)]. As this common domain is important in binding to epithelialcells [see Doig et al., Infection and Immun., 56: 1641-1646 (1988)], itis useful in the production of a broadly effective P. aeruginosa pilivaccine.

The surface of many gram-negative bacteria, e.g., E. coli, P.aeruginosa, M. bovis, N. gonorrhea, are covered with filamentousstructures called pili or fimbriae. Pili are composed primarily ofprotein (pilin) and have been found to act as antigenic determinantswhen injected into test animals. Certain pili, including PAO, PAK, CD4,as they are commonly referred to, and others, mediate the colonizationof P. aeruginosa in humans. Some bacterial cells lacking these pili,either through mutation or loss of the plasmid carrying the pilus gene,are incapable of colonizing mucosa. Apparently, the pili on the surfaceof the bacterium adhere to the lining of the throat and trachea throughspecific interactions with epithelial cell receptors. P. aeruginosa canutilize both pili and alginate (the principle component of the P.aeruginosa capsule) as adhesins to mediate attachment to humanrespiratory epithelial cells [see Doig et al., Infection and Immun., 55:1517-1522 (1987); Doig et al., Infection and Immun. 56: 1641-1646(1988); Marcus et al., Infection and Immun., 47: 723-729 (1985); Ramphalet al., Infection and Immun. 44: 38-40 (1984); Ramphal et al., Infectionand Immun., 47: 1-4 (1985); woods et al., Infection and Immun., 29:1146-1151 (1980).

Equilibrium analysis of P. aeruginosa binding to human respiratoryepithelial cells indicates that the Pseudomonas pilus adhesin has aconsiderably higher apparent affinity or binding constant (Ka) than doesthe alginate adhesin [McEachran et al., Can. J. Microbiol. 31: 563-569(1985), McEachran et al., J. Microbiol. Meth., 5: 99-111 (1986); Doig etal., Infection and Immun., 55: 1517-1522 (1987)].

These observations suggest that the pilus adhesin is likely the dominantPseudomonas adhesin in the initiation of an infection. Adhesin-mediatedanchorage is a prerequisite for the induction of disease by P.aeruginosa.

Anything which would biologically interfere with this adhesion should beeffective in blocking infection. Such a technique has been investigatedby monoclonal antibody treatment of bacterial adhesion and has beenreported in the patent literature, e.g., U.S. Pat. No. 4,443,549 andU.S. Pat. No. 4,702,911 and published U.S. PCT application Ser. No.PCT/US85/00565. It is important to note that bacterial adhesins areunique so that this technique is not predictable with various otherbacteria.

SUMMARY OF THE INVENTION

According to an aspect of the invention, a polypeptide is provided thatis smaller than a naturally-occurring P. aeruginosa pilin protein, andthe pharmaceutically acceptable salts thereof, that are capable ofimmunologically mimicking a conserved antigenic determinant site withina semi-variable region of the carboxy-terminal half of the P. aeruginosapilin and thus are capable of being an antigen, or an immunogen,operable in blocking a P. aeruginosa infection. Additionally, thepresent invention provides a vaccine that contains such an immunogen aswell as a method of immunization against a P. aeruginosa infection. Thisinvention further contemplates a diagnostic assay utilizing apolypeptide of this invention and/or a receptor such as an antibodyelicited by such a polypeptide.

According to an aspect of the invention, the pure homogeneouspolypeptide comprises at least one amino acid residue sequence,containing about 12 amino acid residues and up to about 20 amino acidresidues, that defines a sequence capable of immunologically mimickingan antigenic determinant site of a P. aeruginosa pilin. This amino acidresidue sequence can repeat as a unit one or more times in the samepoly-peptide molecule. More than one type of such repeating unit, andmore than one repeating unit of the same type, can be present in asingle polypeptide molecule that embodies the present invention.

Such a polypeptide can be made as a protein synthesized by geneticengineering techniques or it can be built-up from individual amino acidresidues, or amino acid residue blocks.

According to a preferred aspect of the invention, pure homogeneouspolypeptides embodying this invention can be defined as including theamino acid residue sequence, taken from left to right and in thedirection from the amino-terminus to the carboxy-terminus, of theformula:

    -X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 -X.sub.8 -X.sub.9 -X.sub.10 -X.sub.11 -X.sub.12 -X.sub.13 -X.sub.14 -X.sub.15 -X.sub.16 -X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -

wherein the X designates an amino acid residue or a blank. If a blankoccurs between residues X_(n) and X_(n+2), where n=1 to 18, then Xn isconnected to X_(n+2) through an amide bond (--CONH--). This amide bondresults from the condensation of the α-carboxy-terminus of residueX_(n+2) with the α-amino-terminus of residue X_(n+2).

In the above formula:

X₁ is the amino acid residue Cysteine (C);

X₂ is an amino acid residue from the group Glycine (G), Lysine (K),Serine (S) or is a blank;

X₃ is an amino acid residue from the group Alanine (A) or Isoleucine (I)or is a blank;

X₄ is an amino acid residue from the group Serine (S) or Threonine (T)or is a blank;

X₅ is an amino acid residue from the group Glycine (G), Lysine (K),Serine (S) or is a blank;

X₆ is an amino acid residue from the group Serine (S) or Threonine (T);

X₇ is an amino acid residue from the group Aspartic acid (D), Leucine(L), Asparagine (N), Proline (P) or is a blank;

X₈ is an amino acid from the group Alanine (A), Leucine (L), Valine (V)or is a blank;

X₉ is the amino acid Threonine (T) or is a blank;

X₁₀ is an amino acid from the group Alanine (A), Asparagine (N),Glutamine (Q) or is a blank;

X₁₁ is an amino acid residue from the group Glycine (G), Tryptophan (N)or is a blank;

X₁₂ is an amino acid residue from the group Aspartic acid (D), Glutamicacid (E), Lysine (K);

X₁₃ is an amino acid residue from the group Alanine (A), Glutamic acid(E), Asparagine (N), Proline (P);

X₁₄ is an amino acid residue from the group Lysine (K), Methionine (M),Asparagine (N), Glutamine (Q);

X₁₅ is an amino acid residue from the group Phenylalanine (F), Tyrosine(Y);

X₁₆ is an amino acid residue from the group Alanine (A), Isoleucine (I),Leucine (L), Arginine (R), Threonine (T);

X₁₇ is the amino acid residue Proline (P);

X₁₈ is an amino acid residue from the group Alanine (A), Lysine (K),Asparagine (N), Serine (S);

X₁₉ is an amino acid residue from the group Glycine (G), Asparagine (N),Threonine (T);

X₂₀ is the amino acid Cysteine (C).

Particularly preferred amino acid residue sequences within the abovegroupings taken from left to right, and in the direction from theamino-terminus to the carboxy-terminus, are:

C T S D Q D E Q F I P K G C

C K S T Q D P M F T P K G C

C T S T Q E E M F T P K G C

C T S N A D N K Y L P K T C

C A T T V D A K F R P N G C

C K I T K T P T A W K P N Y A P A N C

C G I T G S P T N W K A N Y A P A N C

C S I S S T L L T G K P N Y A P S N C

The description of the synthetic peptide of this patent does notrepresent a previously published sequence but is a composite of thecritical residues involved in binding to buccal and tracheal epithelialcell surface receptors. Several sequences have been published and aresubsets of this composite sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are discussed with respect to thefollowing Examples in which:

FIG. 1 is a graph showing the binding of synthetic peptide Ac17red andAc17ox to human BECs;

FIG. 2 is a modified Lineweaver-Burk plot of the binding of syntheticpeptides to human BECs;

FIG. 3 is a modified Lineweaver-Burk plot of the binding of PAK pili tohuman BECs;

FIG. 4 is a blot showing the binding of PAK pili and synthetic peptideto blotted BEC proteins on nitrocellulose;

FIG. 5 shows micrographs A, B, C and D of indirect immunofluorescentlocalization of PAK pili binding to fractionated ciliated TECs;

FIG. 6 shows micrographs A, B, C, D, E, and F of indirectimmunofluorescent localization of binding of synthetic peptides to humanciliated TECs; and

FIG. 7 is a bar graph demonstrating the Fab fragments produced againstregions other than the C-terminal of PAK pilin and are ineffective inpreventing pilin binding to BECs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Polypeptides of the present invention are smaller than thenaturally-occurring Pseudomonas aeruginosa pilin protein and include anamino acid residue sequence of about 12 to about 20 amino acid residues,preferably 12 to 20 amino acid residues, that immunologically mimic aconserved antigenic determinant site in the region of thecarboxy-terminal half of P. aeruginosa pilin protein. As such, thepresent polypeptides are useful by themselves, or as pharmaceuticallyacceptable salts, as the active constituent in a vaccine, as aninoculum, or in a diagnostic assay. The polypeptides of this inventionare prepared by a variety of synthetic techniques as will be described.Such synthetic production of the subject polypeptides result in purematerials; i.e., homogeneous peptide sequences which are substantiallyfree of at least any foreign biological materials.

The term "antigenic determinant", as used herein, designates thestructural component of a molecule that is responsible for specificinteraction with corresponding antibody (immunoglobulin) moleculeselicited by the same or related antigen. Antigenic determinants in thepresent polypeptides comprise chemically active surface groupings ofamino acid residues.

The term "antigen", as used herein, means an entity that is bound by anantibody.

The term "immunogen", as used herein, describes an entity that inducesantibody production in the host animal. In some instances the antigenand the immunogen are the same entity, while in other instances the twoentities are different.

The phrase "immunologically mimicks" is used herein to mean that animmunogenic polypeptide of this invention is not a natural protein or acleaved fragment of a natural protein, but a manufactured polypeptide,as by solid phase synthesis or genetic engineering techniques, whichpolypeptide induces production of antibodies that bind to the inducingpolypeptide and also to a corresponding pilin or pilin polypeptideportion.

All amino acid residues identified herein are in the natural orL-configuration unless otherwise specified. In keeping with standardpeptide nomenclature, abbreviations for amino acid residues that havebeen used herein are as follows:

    ______________________________________                                        Symbol                                                                        1 Letter     3 Letter    Amino Acid                                           ______________________________________                                        Y            TYR                                                              L-tyrosine                                                                    G            GLY                                                              glycine                                                                       F            PHE                                                              L-phenylalanine                                                               M            MET                                                              L-methionine                                                                  A            ALA                                                              L-alanine                                                                     S            SER                                                              L-serine                                                                      I            ILE                                                              L-isoleucine                                                                  L            LEU                                                              L-leucine                                                                     T            THR                                                              L-threonine                                                                   V            VAL                                                              L-valine                                                                      P            PRO                                                              L-proline                                                                     K            LYS                                                              L-lysine                                                                      N            ASN                                                              L-asparagine                                                                  H            HIS                                                              L-histidine                                                                   Q            GLN                                                              L-glutamine                                                                   E            GLU                                                              L-glutamic acid                                                               W            TRP                                                              L-tryptophan                                                                  R            ARG                                                              L-arginine                                                                    D            ASP                                                              L-aspartic acid                                                               C            CYS                                                              L-cysteine                                                                    ______________________________________                                    

The term "pharmaceutically acceptable salts", as used herein, refers tothe non-toxic salts, such as, alkali metal, alkaline earth metal andammonium salts commonly used in the pharmaceutical industry includingthe sodium, potassium, lithium, calcium, magnesium, and ammonium saltsand the like which are prepared by methods well known in the art. Theterm also includes non-toxic acid addition salts which are generallyprepared by reacting the compounds of this invention with a suitableorganic or inorganic acid. Representative salts include thehydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,valerate, oleate, laureate, borate, benzoate, lactate, phosphate,tosylate, citrate, maleate, fumarate, succinate, tartrate and the like.The polypeptides meeting the foregoing conditions elicit antibodies in amammalian host and are believed to immunologically mimic a desiredantigenic determinant site within a region of the carboxy-terminal halfof the pilin protein.

One or more amino acid residue sequences meeting the foregoingconditions can be present as repeating units. Additionally, polypeptidescontaining one or more such amino acid residue sequences can be formedinto relatively larger synthetic moieties by joining the individualpolypeptides head-to-tail.

These polypeptides can be characterized as those including the aminoacid sequences taken from left to right in the direction from theamino-terminus to the carboxy-terminus, of the formula:

    -X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X .sub.7 -X.sub.8 -X.sub.9 -X.sub.10 -X.sub.11 -X.sub.12 -X.sub.13 -X.sub.14 -X.sub.15 -X.sub.16 -X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20

wherein the X designates an amino acid residue or a blank. If a blankoccurs between residues X_(n) and X_(n+2), where n=1 to 18, then X_(n)is connected to X_(n+2) through an amide bond (--CONH--). This amidebond results from the condensation of the α-carboxy-terminus of residueX_(n) with the α-amino terminus of residue X_(n+2).

Additionally,

X₁ is the amino acid residue Cysteine (C);

X₂ is an amino acid residue from the group Glycine (G), Lysine (K),Serine (S) or is a blank;

X₃ is an amino acid residue from the group Alanine (A) or Isoleucine (I)or is a blank;

X₄ is an amino acid residue from the group Serine (S) or Threonine (T)or is a blank;

X₅ is an amino acid residue from the group Glycine (G), Lysine (K),Serine (S) or is a blank;

X₆ is an amino acid residue from the group Serine (S) or Threonine (T);

X₇ is an amino acid residue from the group Aspartic acid (D), Leucine(L), Asparagine (N), Proline (P) or is a blank;

X₈ is an amino acid from the group Alanine (A), Leucine (L), Valine (V)or is a blank;

X₉ is an amino acid from the group Threonine (T) or is a blank;

X₁₀ is an amino acid from the group Alanine (A), Asparagine (N),Glutamine (Q) or is a blank;

X₁₁ is an amino acid residue from the group Glycine (G), Tryptophan (N)or is a blank;

X₁₂ is an amino acid residue from the group Aspartic acid (D), Glutamicacid (E), Lysine (K);

X₁₃ is an amino acid residue from the group Alanine (A), Glutamic acid(E), Asparagine (N), Proline (P);

X₁₄ is an amino acid residue from the group Lysine (K), Methionine (M),Asparagine (N), Glutamine (Q);

X₁₅ is an amino acid from the group Phenylalanine (F), Tyrosine (Y);

X₁₆ is an amino acid residue from the group Alanine (A), Isoleucine (I),Leucine (L), Arginine (R), Threonine (T);

X₁₇ is the amino acid residue Proline (P);

X₁₈ is an amino acid residue from the group Alanine (A), Lysine (K),Asparagine (N), Serine (S).

X₁₉ is an amino acid residue from the group Glycine (G), Asparagine (N),Threonine (T).

X₂₀ is the amino acid Cysteine (C).

Particularly preferred amino acid residue sequences within the abovegroupings taken from left to right, and in the direction from theamino-terminus to the carboxy-terminus, are:

C T S D Q D E Q F I P K G C

C K S T Q D P M F T P K G C

C T S T Q E E M F T P K G C

C T S N A D N K Y L P K T C

C A T T V D A K F R P N G C

C K I T K T P T A W K P N Y A P A N C

C G I T G S P T N W K A N Y A P A N C

C S I S S T L L T G K P N Y A P S N C

which sequences have been found to be located in the region of thecarboxy-terminus half of pilin proteins from eight different strains ofP, aeruginosa as described by Pasloske et al., J. of Bacteriology, 170:3738-3741 (1988).

Particularly preferred structures for the sequences described here arethose in which these sequences contain intramolecular disulfide bonds.These disulfide bonds occur through oxidative coupling of the sulphuratoms of the two Cysteine (c) residues contained in each sequence.

In a typical laboratory preparation, 10 milligrams of thedi-CYS-polypeptide (containing amino- and carboxy-terminal cysteineresidues in unoxidized form) are dissolved in 250 milliliters of 0.1molar ammonium bicarbonate buffer having a pH value of about 8. Thedissolved di-CYS-polypeptide is then air oxidized by stirring theresulting solution gently for a period of about 18 hours, or until thereis no detectable free mercaptan by the Ellman test. [See, Ellman, Arch.Biochem. Biophys. 82: 70-77 (1959)]. The cyclized peptide so prepared isthen typically isolated by freeze drying, redissolution andchromatographic purification.

These preferred sequences have been found to be conserved within theregion of the carboxy-terminal half of pilin proteins from eightdifferent strains of P. aeruginosa as described by Pasloske et al., J.Bacteriology 170: 3738-3741 (1988) as well as antigenically relatedvariants thereof as defined herein below.

More than one of the foregoing sequences can be present in the samepolypeptide, usually spaced from one another by a chain of other aminoacid residues or other suitable linking group.

Biochemical evidence from immunoassay and from analogy with conservedprotein-protein interaction in solved X-ray crystallographic structureswith differing sequences such as in the dimer contacts of oligomericenzymes, indicates that the conservation of protein-protein recognitiondoes not require a strict conservation of sequence, for relatedness.While single amino acid residue changes may affect such recognition to awide degree depending upon the nature of the substitution, in generalterms, the relatedness of two differing amino acid sequences withrespect to protein-protein (and antigenic and/or immunogenic)recognition can be expressed in terms of seven basic amino acidparameters:

(1) hydrophobicity;

(2) polarity;

(3) size of side chain;

(4) charge;

(5) preference for turned secondary structure;

(6) preference for beta strand secondary structure; and

(7) preference for helical secondary structure.

To define the degree of sequence identity relevant to antigenic and/orimmunogenic recognition, and thus antigenically related variants, thefollowing classification using empirical similarities between amino acidresidues can be used. This classification is based on an arrangement ofamino acid residues which reveals exchange groups. Amino acids withinsuch a group exchange preferentially with each other. Therefore, theyresemble each other most with respect to their overall impact on proteinstructures. These empirical similarities are outlined in "Principles ofProtein Structure", Charles R. Cauton, Editor, Springer-Verlag, NewYork, Inc. 1979, page 14 and are as follows:

    ______________________________________                                        Exchange Group                                                                           Description                                                        ______________________________________                                        1          The aromatics Phe (F), Tyr                                                    (Y) and Tryptophan (W);                                            2          The positively charged residues Lys (K),                                      Arg (R) and His (H);                                               3          The large aliphatic nonpolar residues Val                                     (V), Leu (L), Ile (I), Met (M) and Cys                                        (C);                                                               4          The small residues Ser (S), Thr (T), Asp                                      (D), Asn (N), Gly (G), Ala (A), Glu (E),                                      Gln (Q) and Pro (P).                                               ______________________________________                                    

For the purpose of this invention a related peptide is defined in thefollowing way:

As any peptide containing a Cysteine residue (C) at position X₁ followedby,

an amino acid residue from groups 2 or 4 in position X₂ or a blank.

an amino acid residue from groups 3 or 4 in position X₃ or a blank.

an amino acid residue from group 4 in position X₄ or a blank.

an amino acid residue from groups 2 or 4 in position X₅ or a blank.

an amino acid residue from group 4 in position X₆ or a blank.

an amino acid residue from groups 3 or 4 in position X₇ or a blank.

an amino acid residue from groups 3 or 4 in position X₈ or a blank.

an amino acid residue from group 4 in position X₉ or a blank.

an amino acid residue from group 4 in position X₁₀ or a blank.

an amino acid residue from groups 1 or 4 in position X₁₁ or a blank.

an amino acid residue from groups 2 or 4 in position X₁₂.

an amino acid residue from group 4 in position X₁₃.

an amino acid residue from groups 2, 3 or 4 in position X₁₄.

an amino acid residue from group 1 in position X₁₅.

an amino acid residue from groups 2, 3 or 4 in position X₁₆.

an amino acid residue from group 4 in position X₁₇.

an amino acid residue from groups 2 or 4 in position X₁₈.

an amino acid residue from group 4 in position X₁₉.

and containing a Cysteine residue (c) at position X₂₀.

The peptides, according to this invention and used herein, arepreferably coupled to higher molecular weight compounds. For example,proteins such as keyhole limpet hemocyanin (KLH) or bovine serum albumin(BSA) or toxoid proteins can be used in the following method.

Peptide Conjugation to Protein Carriers

The peptides were conjugated to keyhole limpet hemocyanin (KLH) orbovine serum albumin BSA via a linker consisting of a [¹⁴ C] glycine anda benzophenone cross-linking group (benzoyl benzoic acid), which wasadded to the peptide during synthesis while the peptide was still on asolid matrix. The hapten was first dissolved in 10-20 μl water in a testtube. The protein carriers (10 mg/100 μl) were then added and mixed.Covalent attachment of the peptide to the carrier occurs followingactivation of the benzoylbenzoyl group by UV irradiation at 4° C. for anhour in a RPR 208 preparative reactor equipped with RPR 3500 Å lamps.Unconjugated hapten were removed by successive dialysis against 8 Murea, 100 mM and 25 mM ammonium bicarbonate. The product wasfreeze-dried and the peptide incorporation determined by measuring theradioactivity incorporated per mole carrier. Peptide/protein ratios ofabout 4:1 and 10:1 were obtained for the oxidized and reduced peptides,respectively.

A mixture of the foregoing polypeptides, including those havingantigenically-related regions as defined herein above, can also be usedto make up a vaccine against or a diagnostic assay for a P. aeruginosainfection, and/or an inoculum for raising antibodies. It is alsounderstood that anti-idiotypic antibodies can be developed as vaccinesbased on the sequence of this invention. Such technique is described inmore detail in Kennedy et al, Vaccines 86 "New Approaches toImmunization", Cold Spring Harbour, 1986, p. 85.

Any interference with bacterial adhesion and subsequent colonizationwill prevent infection. Monoclonal antibodies specific for a portion ofthe amino acid sequence of the P. aeruginosa pili protein can interactwith the adhesins and interfere with cell anchorage. Alternatively, apeptide or portion of the whole pilin amino acid sequence can be used asan immunogen to develop host antibodies which would, in turn, act toprevent bacterial anchorage and subsequent colonization. These methods,therefore, represent new and useful biological agents for the preventionand treatment of P. aeruginosa disease in humans. It should be notedthat this synthetic vaccine is not directed at the dominant immunogenicor antigenic site on P. aeruginosa but rather to the adhesin bindingsite responsible for attachment of the bacterium to the surfaces ofhuman buccal and tracheal epithelial cells. Vaccines containingeffective amounts of the present polypeptides induce production ofantibodies in a sufficient amount to protect the vaccinated individualfrom infection with P. aeruginosa. Booster injections can be given ifneeded.

Thus, the word "vaccine" in its various grammatical forms is used hereinin relation to the protection of a host mammal. The word "inoculum" inits various grammatical forms is used herein to describe a compositioncontaining a polypeptide of this invention as an active ingredient usedfor the preparation of antibodies that immunologically bind to P.aeruginosa pili. A vaccine and an inoculum may thus contain theidentical ingredients, but their uses are different.

The polypeptides suitable as antigens or immunogens, or both, for thepresent purposes can be produced synthetically or by genetic engineeringtechniques, and can be in monomeric as well as multimeric forms for usein vaccines, inocula, or as diagnostics. When used in a vaccine orinoculum, the polypeptide may be used alone, as in the case of anoligomer or a multimer, or used linked to another carrier moiety as aconjugate. When used alone as an immunogen, a polypeptide of thisinvention typically contains from about 12 to about 20 amino acidresidues. Such polypeptides are preferably linked to a carrier. Thepolypeptides of this invention are therefore pure and homogeneouswithout any extraneous matter as would be experienced if thepolypeptides were isolated from naturally occurring polypeptides.

Particularly useful conjugate carriers include keyhole limpet hemocyanin(KLH), tetanus toxoid, poly-L-(LYS:GLU), peanut agglutinin,poly-D-Lysine, diphtheria toxoid, ovalbumin, soybean agglutinin, bovineserum albumin (BSA), human serum albumin, and the like.

The term "manufactured" as used herein means that the polypeptidemolecule or polypeptide repeating unit has been built up syntheticallyby chemical means, i.e., chemically synthesized or by human-mediatedbiological means, e.g., by genetic engineering techniques, which includerecombinant DNA techniques and vaccinia viruses as vectors for vaccineantigens. Such latter technique is disclosed in more detail by GeraldQuinnan, "Proceedings of a Workshop", Nov. 13-14, 1984 Elsevier, p. 27.Thus, the manufactured polypeptides embodying the present invention arefree from naturally occurring proteins and fragments thereof. Thewell-known solid phase chemical synthesis in which blocked amino acidresidues are added in a serial manner to obtain the desired polypeptideis the preferred method of synthesis, and is discussed in greater detailherein below.

As mentioned herein above, polypeptides suitable for the purposes of thepresent invention can be synthesized by the well-known solid phasemethod. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149-2154(1963), Houghten et al., Int. J. Pept. Proc. Res. 16: 311-320 (1980) andParker and Hodges, J. Prot. Chem. 3: 465-478 (1985), for a completediscussion of these techniques. The solid phase method of polypeptidesynthesis can be practiced utilizing a Beckman Model 990B PeptideSynthesizer, available commercially from Beckman Instruments Co.,Berkeley, Calif., U.S.A.

In preparing a synthetic polypeptide of this invention by the abovesolid phase method, the amino acid residues are linked to a resin (solidphase) through an amide linkage from the carboxy-terminal residue.

The alpha-amino group of each added amino acid typically is protected bya tertiary-butoxy-carbonyl (t-Boc) group prior to the amino acid beingadded into the growing polypeptide chain. The t-Boc group is thenremoved prior to addition of the next amino acid to the growingpolypeptide chain. Reactive amino acid side chains are also protectedduring synthesis of the polypeptide. Usual side-chain protecting groupsused for the remaining amino acid residues are as follows:O-(p-bromobenzyoxycarbonyl) for tyrosine, O-benzyl for threonine,serine, aspartic acid and glutamic acid, and S-methoxy-benzyl forcysteine, 2-chlorobenzyloxycarbonyl lysine and formyl tryptophane.Protected amino acids are recrystallized from appropriate solvents togive single spots by thin layer chromatography. Couplings are typicallycarried out using a 2-fold molar excess of both protected amino acid anddicyclohexyl carbodiimide over the number of milli-equivalents ofinitial N-terminal amino acid. For asparagine and glutamine, an equalmolar amount of N-hydroxy-benzotriazole is added to the protected aminoacid and dimethyl-formamide is used as the solvent. All couplingreactions are typically more than 99% complete by the picric acid testof Gisin, Anal. Chem. Acta, 58: 248-249 (1972), or the ninhydrin test,Sarin et al., Anal. Biochem. 117: 147-157 (1981).

A portion of the resulting, protected, resin-bonded polypeptide (1 gram)is treated with two milliliters of anisole, and anhydrous hydrogenfluoride, 20 milliliters, is condensed into the reaction vessel at dryice temperature. The resulting mixture is stirred at 4° for 1.0 hour tocleave the protecting groups and remove the polypeptide from the resin.After evaporating the hydrogen fluoride at a temperature of 4° C. with astream of N₂, the residue is extracted with anhydrous diethyl etherthree times to remove the anisole, and the residue is dried in vacuo.

The vacuum dried material is extracted with neat trifluoro acetic acid(3 times 10 milliliters each). The extraction separates the freepolypeptide from the resin. After dilution with water to a concentrationof 10-20% acetic acid, the resulting solution is lyophilized to providea monomeric, unoxidized, polypeptide. The peptide released from theresin is then purified by known standard chromatographic procedures andthen oxidized to give the monomeric intramolecular cyclized product.

In a typical laboratory preparation, 10 milligrams of the di-Cyspolypeptide (containing amino- and carboxy-terminal cysteine residues inunoxidized form) are dissolved in 250 milliliters of 0.1 molar ammoniumbicarbonate buffer having a pH value of about 8. The dissolved di-Cyspoly-peptide is then air oxidized by stirring the resulting solutiongently for a period of about 18 hours, or until there is no detectablefree mercaptan by the Ellman test [see, Elman, Arch. Biochem. Biophys,82: 70-77 (1959)].

This cyclic oxidized peptide can also be polymerized from head-to-tailusing the following procedure in which the cyclic polypeptide isdissolved in dimethylformamide (1 mg/mL) and benzotiazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (1.1 molar equivalents)and diisopropylethylamine (100 μL) are added and allowed to react atroom temperature for about eight hours. The polymeric peptides producedin this fashion are isolated by evaporation of the solvents andchromatographic separation. Benzyl protecting groups are then removed bytreatment of the polymer with anisole and anhydrous hydrogen fluoride at-10° C. for 110 hour. After removal of the hydrogen fluoride at -10° C.with a stream of N₂ the residue is extracted with anhydrous diethylether three times to remove the anisole and the residue. Polypeptidemultimers containing oxidized cyclic monomeric units may be used for thepreparation of vaccine against Pseudomonas aeruginosa. In this event themethod of synthesis must follow the known solid-phase method developedby Atherton et al., J. C. S. Perkin 1: 538-546 (1981), which disclosuresare incorporated herein by reference. This method can be practicedutilizing an LKB BioLynx model 4175 peptide synthesizer availablecommercially from LKB Biochrom, Ltd., Cambridge, England.

In preparing a synthetic polypeptide of this invention by the abovesolid-phase method, the amino acid residues are linked to a resin(solid-phase) through an ester linkage from the carboxy-terminalresidue.

The alpha-amino group of each added amino acid typically is protected bya 9-fluoroenyl-methoxycarbonyl (FMOC) group prior to the amino acidbeing added onto the growing polypeptide chain. The FMOC group is thenremoved prior to addition of the next amino acid to the growingpolypeptide chain. Reactive amino acid side chains are also protectedduring synthesis of the polypeptide. Usual side-chain protecting groupsused for the remaining amino acid residues are as follows:o-(p-bromobenzoyloxycarbonyl) for tyrosine, o-benzyl for threonine andserine, -phenacyl for aspartic and benzyl for glutamic acid,S-tert-butyl for cysteine and 2-chlorobenzyloxy-carbonyl for lysine.Couplings are typically carried out using a 2-fold molar excess ofprotected amino acid and one equivalent of dicyclohexyl carbodiimideover the number of milliequivalents of initial N-terminal amino acid.For asparagine (N) and glutamine (Q), 2 molar equivalents ofN-hydroxy-benzotriazole and dicyclohexyl carbodiimide are used. Allcoupling reactions are typically monitored by the ninhydrin test ofSarin, Anal-Biochem. 117: 147-157 (1981) and are typically more than 99%complete.

A portion of this resin is cleaved to release peptide for oxidation andpolymerization. Cleavage to release partially deprotected peptide isaccomplished using the following method which utilized 95%trifluoroacetic acid (5 mL/50 mg resin). The resin (50 mg) is suspendedin 95% trifluoroacetic acid containing anisole (3%), ethandithiol (1%)and ethylmethyl sulphide (1%) by volume and the reaction is allowed toproceed at room temperature for 2-3 hours. The resin is filtered toremove the peptide and scavengers and the resin is washed with neattrifluoroacetic acid (3-5 mL). The combined filtrates are evaporatedunder vacuum and the residue triturated with diethyl ether. Finally theresidue is dissolved in 0.5% trifluoroacetic acid in water andlyophilized.

This crude peptide can be purified by known reversed-phase HPLC methodsand the purified peptide cyclized by air oxidation in 0.1 molar ammoniumbicarbonate buffer having a pH value of about 8 for a period of about 18hours as described hereinabove. Isolation of the cyclized product fromthe oxidation procedure follows known chromatographic techniques.Synthesis of the polymeric polypeptide is performed by head-to-tailcoupling of the free amino and carboxy-termini usingbenzotriazol-1-yloxytris (dimethyl amino) phosphoniumhexafluorophosphate (1.1 molar equivalent) and diisopropyl ethylamine(100 1) at room temperature for about 8 hours. Purification of themultimers is then accomplished using known size-exclusionchromatographic techniques. Cleavage of the remaining benzyl side chainprotecting groups is carried out by treatment of the multimers withanhydrous hydrogen fluoride (9 ml) containing anisole (1 mL) for about 1hour at about -10° C. Isolation of the deprotected multimers isperformed by evaporation of the hydrogen fluoride under a stream of N₂gas while maintaining the temperature at about -10° C. The product isthen triturated with diethyl ether to remove the scavenger and dissolvedin aqueous 0.5% trifluoroacetic acid and lyophilized.

Alternatively, if the sequence contains benzyl protected serine andthreonine in combination with tert-butyl cysteine, then cleavage beforecyclization may be carried out in liquid anhydrous hydrogen fluoride (9mL) containing anisole (1 mL) and ethandithiol (25 μL). Subsequent tothis cleavage, cyclization and polymerization will provide themultimeric polypeptide. Purification at each step in the synthesis isaccomplished utilizing chromatographic techniques known in the art.

Alternatively, purified cyclic polypeptide obtained by the methodsdescribed hereinabove may be attached directly to a core matrix calledthe multiple antigen peptide system (MAP) known in the art and describedby J. P. Tam, Proc. Natl. Acad. Sci. U.S.A. 85: 5409-5413 (1988).Attachment of cyclic monomeric polypeptide is performed using an 8:1molar ratio of polypeptide to core matrix in dimethylformamidecontaining 8 equivalents of benzotriazol-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate and diisopropylethylamine (100 μL).

Vaccines and inocula of the present invention may be administered byinjection, usually intramuscularly or subcutaneously, orally by means ofan enteric capsule or tablet, as a suppository, as a nasal spray, and byother suitable routes of administration. For a human patient, a suitabledose of the polypeptide depends, in part, upon the chosen route ofadministration and a number of other factors. Included among thosefactors are the body weight of the mammal to be immunized, the carrierwhen used, the adjuvant when used, and the number of inoculationsdesired to be used.

Individual inoculations for a human patient typically contain unit dosesof about 10 micrograms to about 100 milligrams of polypeptide, exclusiveof any carrier to which the polypeptide may be linked. If desired, aseries of doses may be administered over a period of time for optimumimmunity. Unit dosage forms of the vaccine can also be provided, ifdesired, containing the aforementioned amounts of the polypeptide.

In any event, the immunogen contained in a vaccine or an inoculum ispresent in an "effective amount", which amount depends upon a variety offactors as is well known in the immunological arts, e.g., the bodyweight of the mammal to be immunized, the carrier moiety used, theadjuvant used, the duration of protection sought, and the desiredimmunization protocol.

Whole antibodies, as well as substantially whole antibodies, raised tothe polypeptides of this invention and antibody combining sites preparedfrom such antibodies constitute still another aspect of this invention.These molecules are collectively referred to as receptors.

Receptors are raised in mammals such as rabbits, goats, horses and thelike by immunization using the inocula described hereinbefore.Immunization procedures are substantially the same as those used invaccinations except that powerful adjuvants, such as Complete FreundsAdjuvant (CFA) and/or Incomplete Freunds Adjuvant (IFA) as they arecommonly known, that are not acceptable for human use can be included inanimal inocula.

Typical inoculum stock solutions are prepared with CFA, IFA or alum asfollows: An amount of the polypeptide, synthetic polypeptide-conjugateor polymeric polypeptide sufficient to provide the desired, effectiveamount of polypeptide per inoculation is dissolved in PBS at a pH valueof 7.2. Equal volumes of CFA or IFA are then mixed with the polypeptidesolution to provide an inoculum containing polypeptide, water andadjuvant in which the water-to-oil ratio is about 1:1. The mixture isthereafter homogenized to provide the inoculum stock solution. When alumis used, about 200 micrograms of conjugate is absorbed onto about 4milligrams of alum to prepare the stock inoculum.

Rabbits can be utilized herein to raise antipolypeptide antibodies. Whenso used, the host rabbit is typically injected subcutaneously with aninoculum comprising 200 micrograms of a polypeptide conjugate(polypeptide plus carrier) emulsified in CFA; 200 micrograms ofpolypeptide conjugate in IFA; and 200 micrograms of polypeptideconjugate with 4 milligrams alum injected intraperitoneally on days 0,14 and 21, respectively, of the immunization schedule. Each inoculation(immunization) consists of four injections of the inoculum. Mice may beimmunized in a similar way using about one tenth of the above dose perinjection.

Animals are typically bled 4 and 15 weeks after the first injection.Control pre-immune serum is obtained from each animal by bleeding justbefore the initial immunization,

Control inoculum stock solutions can also be prepared with keyholelimpet hemocyanin (KLH), KLH in CFA or IFA, KLH-alum absorbed, KLH-alumabsorbed-pertussis, edestin, thyroglobulin, tetanus toxoid, tetanustoxoid in IFA, cholera toxoid and cholera toxoid in IFA, and the like.

The efficacy of the above immunization procedure is typically determinedby means of an ELISA in which the immunogenic polypeptide of thisinvention is used as the antigen to determine the amount of antibodiespresent in diluted sera obtained from the above bleeds. Sera thatprovide anti-polypeptide antibody titers (dilutions) of at least about1:160 are considered useful in providing the antibodies of thisinvention. The typically utilized ELISA test is described in greaterdetail in Bittle et al., Nature 298, 30-33 (1982).

Suitable monoclonal receptors, typically whole antibodies, may also beprepared using hybridoma technology as described by Niman et al., Proc.Natl. Acad. Sci. USA 80: 4949-4953 (1983). Monoclonal receptors need notonly be obtained from hybridoma supernatants, but may also be obtainedin generally large quantities from ascites fluid of mammals into whichthe desired hybridoma has been introduced. Production of monoclonalantibodies using ascites fluid is well known and will not be dealt withfurther herein.

A receptor of this invention binds both to the polypeptide to which itwas raised and also to the corresponding pilin protein whose antigenicdeterminant site the polypeptide of this invention immunologicallymimicks. Thus, a polypeptide of this invention may be both an immunogenand an antigen.

The receptors of this invention are a subset of the naturally occurringpolyclonal antibodies since they are raised to an immunogen which mimicsa small fragment of an intact pilin molecule. Consequently, receptors ofthis invention bind to epitopes of the polypeptide (which is a part ofthe pilin molecule) while naturally occurring antibodies raised to aPseudomonas pilin bind to epitopes throughout the pilin molecule.

The polypeptides, antibodies, and antibody combining sites provided bythese polypeptides, and methods of the present invention may also beused for diagnostic tests, such as immunoassays. Such diagnostictechniques include, for example, enzyme immune assay, enzyme multipliedimmunoassay technique (EMIT) enzyme-linked immunosorbent assay (ELISA),radio-immune assay (RIA), fluorescence immune assay, either single ordouble antibody techniques, and other techniques in which either theantibody combining site or the antigen is labeled with some detectabletag. See generally Maggio, Enzyme Immunoassay, CRC Press, Cleveland,Ohio (1981) and Goldman, M., Fluorescent Antibody Methods, AcademicPress, New York, N.Y. (1980).

An illustrative diagnostic system embodying the present invention todetect P. aeruginosa contains receptor molecules such as antibodies,substantially whole antibodies, or antibody combining sites, raised to apolypeptide of this invention. The system also includes an indicatingmeans for signaling the presence of an immunoreaction between thereceptor and the antigen. The indicating means allows the immunoreactionto be detected. When mixed with a body sample such as sputum, thereceptor molecule immunoreacts with the pilin antigen to form animmunoreactant, and the indicating means present then signals theimmunoreaction.

One such exemplary embodiment is an immunofluorescent assay in which asputum smear is acetone-fixed to a plain microscope slide. An aliquot ofantibodies raised in accordance with this invention, e.g., raised inrabbits, generally about 10 micrograms to about 500 micrograms, isincubated on the slides using well-known techniques.

After rinsing away an unimmunoreacted antibodies and blockingnon-specific binding sites on the slide with a protein such as BSA, asecond antibody, such as a goat anti-rabbit antibody can then beincubated on the test slide, if desired. The second antibody is labeledby being linked to a fluorochrome dye such as fluorscein isothiocyanate(FITC).

After this second incubation, any excess of the second antibody isrinsed off leaving any FITC-labeled goat anti-rabbit antibodies thatbound to the first antibodies on the test slide. Presence of theFITC-labeled antibodies may be detected using fluorescent microscopy andthereby signal the presence of a Pseudomonas infection.

The use of whole, intact, biologically active antibodies for thereceptor molecules is not necessary in many diagnostic systems such asthe immunofluorescent assay described above. Rather, only theimmunologically active, idiotype-containing, antigen binding andrecognition receptor site; i.e., the antibody combining site, of theantibody molecule may be used. Examples of such antibody combining sitesare those known in the art as Fab and F(ab')₂ antibody portions that areprepared by methods well known in the art.

Another diagnostic method of this invention is an ELISA assay. Here, apolypeptide antigen of this invention is bound on a solid support suchas the walls of a microtiter plate. Non-specific binding sites on themicrotiter well walls are thereafter blocked with a protein such as BSA.Unbound polypeptide and BSA are removed from the microtiter well as byrinsing.

A body sample such as that above is admixed with an excess of anantibody of this invention in an aqueous solution, and the admixture ismaintained for a time sufficient to form an immunoreaction between theantibody and any Pseudomonas pili antigen present. That liquid admixtureis then mixed with the above-described polypeptide-bound solid supportto form a second admixture containing solid and liquid phases. Thesolid/liquid phase admixture is maintained for a time sufficient forpreviously unreacted antibodies to immunoreact with the polypeptideantigen. The liquid phase is thereafter separated from the solid phase.A solution of a second, labeled antibody that reacts with thefirst-named antibody is then admixed with the solid phase. An exemplarysecond antibody is an alkaline-phosphatase-linked goat anti-rabbit IgGwhere the first-named antibodies are raised in rabbits. The admixtureformed from the solid phase and the second, labeled antibody solution ismaintained for a time period sufficient to form an immunoreactionbetween the two antibodies. The solid and liquid phases are thereafterseparated.

A solution containing a substrate for the enzyme such asp-nitrophenylphosphate is thereafter admixed with the solid phase. Theoptical density at a preselected wave length (e.g., 405 nanometers) maythen be determined after a predetermined time period has elapsed andcompared to the optical density of a control to determine whether thePseudomonas antigen was present in the body sample.

The present invention is further illustrated by the following detailedexamples.

EXAMPLE 1 Polypeptide Synthesis

A series of short synthetic polypeptides whose amino acid residuesequences correspond to small segments of the Pseudomonas pilin proteinwere synthesized according to the method of Merrifield, J. Am. Chem,Soc. 85: 2149-2154 (1963), as modified by Houghten et al., Int. J. Pept.Proc. Res. 16: 311-320 (1980), using a. Beckman Model 990B PeptideSynthesizer (Beckman Instruments Co., Berkeley, Calif., U.S.A.). Thepolypeptide designations and the location in the Pseudomonas pilinprotein of the corresponding amino acid sequence is shown in Table 1,below.

                  TABLE 1                                                         ______________________________________                                        Synthetic Polypeptide Corresponding to                                        Pseudomonas Pilin Segment                                                     Designation.sup.1                                                                      Location.sup.2                                                                         Amino Acid Residue Sequence                                 ______________________________________                                        PAK 128-144                                                                            128-144  K C T S D Q D E Q F I P K G C S                             ______________________________________                                         .sup.1 Polypeptide coupled to KLH or BSA using the benzophenone               crosslinking group as described in Example 2.                                 .sup.2 Location corresponds to the amino acid residue positions of the        Pseudomonas pilin protein sequence described by Sastry et al., FEBS Lett.     (1983) 151: 253-256.                                                     

EXAMPLE 2 Polypeptide-Carrier Couplings

The peptides were conjugated to keyhole limpet hemocyanin at bovineserum albumin via a linker consisting of a norleucine spacer and abenzophenone cross-linking group (benzoyl benzoic acid), which was addedto the peptide during synthesis while the peptide was still on the solidmatrix. The protein (˜3 mg) was first dissolved in 10 to 20 μL of waterin a test tube. The protein carriers (10 mg/100 μL) were then added andmixed. Covalent attachment of the peptide to the carrier occurredfollowing activation of the benzoylbenzoyl group by UV irradiation at 4°C. for 1 hour in a RPR 208 preparative reactor (Rayonet, The SouthernNew England Ultraviolet Co., Middletown, Conn.) equipped with RPR-350 mmlamps. Unconjugated haptens were removed by successive dialysis against8 M urea, 10 mM ammonium bicarbonate, and 25 mM ammonium bicarbonate.The product was freeze-dried. The peptide incorporation was determinedby hydrolysis of a small sample of the conjugate and calculating theratio of the residue norleucine with respect to any reference amino acidnot found in the peptide sequence but contained in the sequence of thecarrier molecule. This ratio then represents the molar ratio ofpeptide:carrier. Peptide/carrier ratios of about 4:1 and 10:1 wereobtained for the oxidized and reduced peptides, respectively.

EXAMPLE 3 Screening of Rabbit Sera for Anti-Polypeptide Antibodies

Rabbit anti-sera were screened for the presence of anti-polypeptideantibodies using an enzyme linked immunosorbent assay (ELISA).Polypeptide antigen made as described in Example 1, above, was adsorbedonto the walls of microtiter plate wells to provide solid phase boundtarget antigen.

A solution of conjugate (5 μg/mL) dissolved in coating buffer, sodiumcarbonate, pH 9.6, was used to coat the wells of a microtiter plate. Theindividual wells were coated with 120 μL of this solution in ahumidified chamber at 4° C. for 16 hours. These plates were then washedwith phosphate buffer saline/Tween three times.

Non-specific binding sites on the microtiter well walls were thereafterblocked by incubating 50 μL of 3% (w/v) BSA/PBS in each well for 4 hoursat 37° C. in a humidified chamber. After incubation, excess BSA wasremoved by inverting and shaking the plates. Polypeptide bound to asolid support whose non-specific binding sites had been blocked was thusprovided for use as target antigen.

To assay the rabbit sera for the presence of anti-polypeptideantibodies, an aliquot of each serum was serially diluted ten-fold in 1%(w/v) BSA/PBS. One hundred microliters of each dilution was contactedwith solid phase bound polypeptide by a mixture in the appropriatemicrotiter wells prepared above. Contact was maintained by incubatingthe wells for about 2 hours at 37° C. in a humidified chamber, thusallowing any anti-polypeptide antibodies present in the serum dilutionsto immunoreact with solid phase bound polypeptide target antigen. Afterincubation, the solid and liquid phases were separated by filling thewells with phosphate buffered saline/Tween inverting and shaking 3 timesin seriatim.

To detect the presence of an immunoreaction between anti-polypeptideantibodies and solid-phase antigen, 100 μL of a 1:1000 dilution of goatanti-rabbit IgG labelled with alkaline phosphatase (Boehringer-MannheimBiochemicals, Indianapolis, Ind.) in phosphate buffered saline/Tween,was added to each well and incubated for about 2 hours in a humidifiedchamber at room temperature. The wells were washed three times withphosphate buffered saline/Tween in seriatim. A substrate solution (50μL) containing 1 mg of p-nitrophenylphosphate in 1 mL of 10%diethanolamine, pH 9.8, was added to each well and the reaction wasallowed to continue for about 45 to 60 min. at room temperature. Theamount of indicating reaction (color development) was quantitated bymeasuring the absorbance of each well at 405 nm. Rabbit antiserademonstrating an absorbance which was 0.05 units above background weredetermined and the results are tabulated in Table 2.

                  TABLE 2                                                         ______________________________________                                        End Point Titers - Direct Elisa Using                                         Rabbit Antisera Against Peptide Conjugate                                     Designation.sup.1                                                                          Peptide-Conjugate Titer                                          ______________________________________                                        17-Rl        3.5 × 10-6                                                 17-R2        7.1 × 10-6                                                 17-01        4.5 × 10-6                                                 17-02        2.1 × 10-6                                                 ______________________________________                                         .sup.1 Antisera derived from 4 different rabbits.                        

EXAMPLE 4 Screening of Rabbit Sera for Anti-Pilin Antibodies

Rabbit antisera were screened for binding to Pseudomonas pili isolatedfrom two strains, PAK and PAO, using direct enzyme-linkedimmunoabsorbent assay (ELISA). The pili were isolated and purified asdescribed by Paranchych et al., Can. J. Microbiol. (1979) 25: 1175-1181and described in Example 5. A 5 μg/mL solution of pili dissolved incoating buffer sodium carbonate, pH 9.6, was used to coat the wells of amicrotiter plate. The individual wells were coated with 120 μL of thissolution in a humidified chamber at 4° C. for 16 hours. These plateswere then washed with phosphate buffered saline/Tween three times.

Non-specific binding sites on the microtiter well walls were thereafterblocked by incubating 50 μL of 3% (w/v) BSA/PBS in each well for 4 hoursat 37° C. in a humidified chamber. After incubation, excess BSA wasremoved by inverting and shaking the plates. Polypeptide bound to asolid support whose non-specific binding sites had been blocked was thusprovided for use as target antigen.

To assay the rabbit sera for the presence of anti-polypeptideantibodies, an aliquot of each serum was serially diluted 10-fold in 1%(w/v) BSA/PBS. One hundred microliters of each dilution was contactedwith solid phase bound polypeptide by a mixture in the appropriatemicrotiter wells prepared above. Contact was maintained by incubatingthe wells for about 2 hours at 37° C. in a humidified chamber, thusallowing any anti-polypeptide antibodies present in the serum dilutionsto immunoreact with solid phase bound polypeptide target antigen. Afterincubation, the solid and liquid phases were separated by filling thewells with phosphate buffered saline/Tween, inverting and shaking 3times in seriatim.

To detect the presence of an immunoreaction between anti- antibodies andsolid-phase antigen, 100 L, of a 1:1000 dilution of goat anti-rabbit IgGlabelled with alkaline phosphatase (Boehringer-Mannheim Biochemicals,Indianapolis, Ind.) in phosphate buffered saline/Tween, was added toeach well and incubated for about 2 hours in a humidified chamber atroom temperature. The wells were washed three times with phosphatebuffered saline/Tween in seriatim. A substrate solution (50 μL)containing 1 mg of p-nitrophenylphosphate in 1 mL of 10% diethanolamine,pH 9.8, was added to each well and the reaction was allowed to continuefor about 45 to 60 min at room temperature. The amount of indicatingreaction (color development) was quantitated by measuring the absorbanceof each well at 405 nm. Rabbit antisera demonstrating an absorbancewhich was 0.05 units above background were determined and the resultsare tabulated in Table 3.

                  TABLE 3                                                         ______________________________________                                        End Point Titers (SD) (n = 3) - Direct                                        ELISA Using Rabbit Antisera Against                                           Pili from Strains PAK and PAO                                                             Pili Titer                                                        Designation.sup.1                                                                           PAK        PAO                                                  ______________________________________                                        17-R1         1.0 × 10.sup.-5                                                                    4.15 × 10.sup.-4                                             (1.2 × 10.sup.-5)                                                                  (3.7 × 10.sup.-4)                              17-R2         1.6 × 10.sup.-5                                                                    4.5 × 10.sup.-4                                              (0.4 × 10-5)                                                                       (0.3 × 10-4)                                   17-01         1.5 × 10.sup.-5                                                                    2.0 × 10.sup.-5                                              (0.7 × 10.sup.-5)                                                                  (0.8 × 10.sup.-5)                              17-02         2.0 × 10.sup.-5                                                                    1.3 × 10.sup.-4                                              (1.2 × 10.sup.-5)                                                                  (1.6 × 10.sup.-4)                              ______________________________________                                         .sup.1 Antisera derived from 4 different rabbits.                             2 The endpoint (n = 3) was determined as the cutoff at an A405 of 0.05 AU                                                                              

EXAMPLE 5 Purification of Pili

The purification procedure used was previously described by Paranchychet al., Can. J. Microbiol. (1979) 25: 1175-1181. Bacteria were grown onsolid medium in large pans as described above and then harvested byscraping the surface of the agar and suspending the cells from 36 trays(about 100 g wet weight) in 1000 mL SSC buffer. The cells were thenstirred with a magnetic stirrer at 5° C. for 2 h. Large bits of agarwere removed by passing the suspension through a sieve and the pili wereremoved from the cells by blending in 200 mL portions for 2 min at 2000rpm with a Sorvall Omnimixer. After removing bacteria by centrifugationat 10,000×g for 15 min. the NaCl concentration of the supernatantsolution was adjusted to 0.5 M. Polyethylene glycol 6000 (PEG 6000) wasthen added to a final concentration of 1% w/v, and the solution wasallowed to sit for 18 h at 4° C. Both pili and flagella precipitatedunder these conditions and were removed by centrifugation at 7000×g for20 min. To remove flagella, the pellet was resuspended in a 10% w/v(NH₄)₂ SO₄ solution (pH 4.0) and allowed to stand at 4° C. for 2 h. Piliprecipitated under these conditions while flagella remained insuspension. Remaining flagella were removed by repeating the ammoniumsulfate precipitation step. The final pellet was redissolved in water,dialyzed exhaustively to remove (NH₄)₂ SO₄, then subjected to CsCldensity gradient centrifugation. The latter procedure involved layering20 mL of pili solution onto 16 mL of a preformed step gradient in whichthe CsCl density ranged from 1.1 to 1.5. After 20 h of centrifugation at20,000 rpm in an SW27 rotor using a Beckman L2-65B ultracentrifuge, thepilus band (buoyant density of about 1.3 g/cm³) was removed, thensubjected to a second CsCl density gradient centrifugation step. Afterremoving the pilus band from the second CsCl gradient and dialyzing toremove CsCl, the pili were resuspended in distilled water and washed byrepeated centrifugation for 2 h at 50,000 rpm in a 60-Ti fixed-anglerotor. The pili were judged pure when SDS polyacrylamide gelelectrophoretic examination of the preparation showed a single proteinband of heavily overloaded samples (100 μg pilin per sample).

EXAMPLE 6 Western Blot Assay

The rabbit antisera were further screened to determine their ability toimmunoreact with Pseudomonas pilin protein in a Western blot assay.Pseudomonas pilin protein was isolated from strains PAK and PAO asdescribed in Example 5. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis was carried out according to the Laemmli method by using15% polyacrylamide running gels. Purified pili (5 μg) and whole-celllysate (3×10⁷ bacteria) were used. The whole-cell lysate was prepared byboiling the PAK cells for 2 to 3 min in 100 μL of sample buffer (2.5%sodium dodecyl sulfate in 0.25 M Tris, pH 6.8) used to dissolve proteinsto be loaded onto the sodium dodecyl sulfate-polyacrylamide gel. Themixture was then centrifuged on a bench-top Microfuge; 10 μL of thesupernatant was removed and diluted with 15 μL of sample buffer beforebeing loaded onto the gel. After separation, the proteins weretransblotted at 0.2 A for 2 to 6 h onto nitrocellulose paper accordingto the method of Towbin et al., P.N.A.S. (USA) (1979) 76: 4350-4354.Excess protein-binding capacity of the nitrocellulose sheet was blockedwith 5% gelatin solution (59 gelatin in 100 mL Tris buffered saline, 20mM Tris, 500 mM NaCl, pH 7.5) for 1 hour at room temperature. Thenitrocellulose sheet was then washed twice with 0.05% Tween-20 in Trisbuffered saline. The sheet was then treated with antipeptide antisera(1:250) diluted with 1% gelatin in Tris buffered saline containingTween-20 at room temperature for 16 hours. Excess sera was removed bywashing twice with Tris buffered saline containing Tween-20. The pilinbands were detected with an immunoassay kit (BioRad Laboratories,Richmond, Calif.) by using a goat anti-rabbit IgG alkaline phosphataseconjugate diluted 3000-fold with 1% gelatin in Tris buffered salinecontaining Tween-20. Incubation with this conjugate was carried out atroom temperature for i hour. Excess conjugate was removed from the sheetby two washes with Tris buffered saline containing Tween-20 followed byone wash with Tris buffered saline alone. Immunoreactants were detectedusing p-Nitro blue Tetrazolium chloride in the presence of5-Bromo-4-chloro-3-indolyl phosphate toluidine salt. Immunoreactantsappear as purple red bands. The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Results of Immunoblot Analysis of PAK and                                     PAO Pilin Proteins with Peptide-Conjugate                                     Rabbit Antisera                                                                              Pilin                                                          Designation.sup.1                                                                              PAK      PAO                                                 ______________________________________                                        17-R1            .sup. +++.sup.2                                                                        +                                                   17-R2            +++      +                                                   17-01            +++      ++                                                  17-02            +++      +                                                   ______________________________________                                         .sup.1 Antisera derived from 4 different rabbits.                             .sup.2 +++, intense band; ++, moderately intense band; +, weak band.     

EXAMPLE 7 Buccal Epithelial Cell Preparation

BEC's were collected from ten healthy non-smoking male volunteers viawooden application sticks rubbed gently on the inside of cheeks, threewooden application sticks per cheek. These sticks were rubbed gentlytogether in 30 mL 10 mM phosphate buffered saline to suspend the BEC's.These cells were washed three times with 30 mL phosphate buffered salineby successive centrifugation (650×g spins) and resuspended. The finalpellet was suspended in 5 mL 10mM phosphate buffered saline at pH 7.2.This suspension was filtered (prewetted 70 μm nylon mesh) and the cellswere diluted to a final concentration of 2×10⁵ cells/mL in phosphatebuffered saline at pH 7.2. This suspension is stored at 4° C. untilready for use.

EXAMPLE 8 Tracheal Epithelial Cell Preparation

Human ciliated tracheal epithelial cells (TECs) were obtained frompatients in the Surgical Intensive care unit at Toronto General Hospitalby bronchoscopic brushing of the bronchial mucosa as described byFranklin et al., Infection and Immunity (1987) 55, 1523-1525.

TECs were obtained by bronchoscopy from surgical patients (under generalanesthetic), intubated intensive care unit (ICU) patients, and healthvolunteers. For the surgical and ICU patients, bronchoscopy wasperformed with a flexible Olympus Type 2 BF bronchoscope insertedthrough an endotracheal tube. A cytology brush was used to abrade thetracheal-bronchial mucosa, and TECs were collected in high-glucoseDulbecco modified Eagle medium containing 1% sodium citrate.

The cell suspension obtained by bronchoscopy contained both ciliated andnonciliated cuboidal and columnar epithelial cells in addition tovarious amounts of mucus, erythrocytes, granulocytes, and cell debrisand was not suitable for direct use in an adhesion assay. The cellsuspension was vortexed briefly, sequentially passed through 70 and 30μm-pore-size-mesh nylon screens, washed twice (500×g for 15 min at 4°C.) with 10 L of 0.01 M phosphate-buffered saline (pH 7.2) (PBS), andthen resuspended in 1 ml of PBS. The cell suspension was thenfractionated by density gradient centrifugation (500×g for 15 min at 4°C. in a swinging bucket rotor) on a PBS-preformed (48,000×g for 40 minat 4° C.) 65% (vol/vol) percoll gradient. The TEC band was collected andapplied to a second percoll gradient.

The ciliated TEC band was collected from the second gradient, and thecells were washed once in PBS and then resuspended in 1.5 ml of PBS. Adirect cell count was performed with a hemacytometer; cell viability wasdetermined by trypan blue dye exclusion. The cell fractionationprocedure typically yielded (2.08±0.34) ×10⁵ cells (mean ± standarderror), of which 32.8±6.5% were ciliated TECs. The vast majority ofthese cells were viable, and in many cases the cilia were still beating.The fractionated TECs contained only epithelial cells, were essentiallyfree of contaminating mucus, and were used directly for adhesion assays.

EXAMPLE 9 Pak 128-144 Binding to BECs

An immunoassay was performed to assess the binding of PAK 128-144 redand PAK 128-144 ox to BECs. BECs (0.2 ml at 2.0×10⁵ BECs/ml) were addedto an equal volume of synthetic peptide (0 nmol/ml to 120 nmol/ml) inPBS and incubated at 37° C. and agitated at 300 rpm. After 1 h, BECswere collected by centrifugation (13,000×g for 2 min at roomtemperature) and washed 5 times with PBS. Monoclonal antibody PK99H (0.2ml of a 10⁻³) dilution in PBS of purified IgG with a titre of 10⁶) wasadded to the BEC pellet and incubated as described above for 1 h. TheBECs were then collected by centrifugation (13,000×g for 2 min at roomtemperature) and washed 5 times with PBS. Goat anti-mouse IgG (H+L)immunoglobin G-horseradish peroxidase conjugate (Jackson Laboratories)was added to the BEC pellet (0.2 ml of a 1:10000 dilution in PBS) andthe mixture incubated as described above for 1 h. The BECs werecollected by centrifugation (13,000×g for 2 min at room temperature) andwashed 5 times with PBS. The pellet was resuspended in 0.2 ml of 1 mMABTS (2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)) in 10 mMcitrate buffer pH 4.2+0.03% peroxide and transferred to a clean tube.The horseradish peroxidase enzyme reaction was stopped by the additionof 0.2 ml of 4 mM NaN₃ and the optical density at 405 nm was determinedafter removal of the BECs by centrifugation. The BEC concentration ineach tube was determined with a hemocytometer at the end of the assayprior to the removal of BECs by centrifugation. Results are shown inFIG. 1 wherein binding of synthetic peptide Ac17red (▪) and syntheticpeptide Ac17ox (+) to human BECs is plotted. Binding of the syntheticpeptides to BECs was determined by a whole cell ELISA assay utilizingthe monoclonal antibody PK99H (which binds to both Ac17px and Ac17red)to quantitate the amount of synthetic peptide bound to the surface ofBECs.

EXAMPLE 10 Pak 128-144 Inhibition of Pilus Binding to BECs

An immunoassay was performed to assess the effect of PAK 128-144 red onpilus binding to BECs. BECs (0.2 ml at 2.0×10⁵ BEC/ml) and syntheticpeptide PAK 128-144 red (0.1 ml such that a final concentration of0,40,80 or 120 nmol/ml of synthetic peptide was obtained) werepreincubated for 30 min at room temperature. Pili (0.1 ml of 0 μg/ml to100 μg/ml) were then added to the BECs with varying concentrations ofsynthetic peptide (0, 40, 80 for 120 nmol/ml). The mixtures were thenincubated for 2 h at 37° C. while being agitated at 300 rpm. BECs andbound pili were then collected by centrifugation (13,000×g for 2 min)and washed 5 times with PBS to remove unbound pili. Monoclonal antibodyPK3B (0.1 ml of a 10⁻⁴ dilution in PBS) (this antibody recognizes PAKpili, but does not react with synthetic peptide PAK 128-144) was thenadded to the BECs with bound pili and incubated for 1 h as describedabove. The remainder of the immunoassay was the same as described forpilus binding to BECs. Results are shown in FIG. 2 wherein a modifiedLineweaver-Burk plot of the binding of synthetic peptides Ac17red (▪)and Ac17ox (+) to human BECs is plotted, and in FIG. 3, wherein amodified Lineweaver-Burk plot of the binding of PAK pili to human BECsin the presence of 0 (X), 40 (X), 80 (Δ), and 120 () nmoles/ml ofsynthetic peptide Ac17red are plotted.

EXAMPLE 11 Pili and Pak 128-144 Binding to TECs

TECs (0.1 ml of 1×10⁵ cells/ml) were mixed with an equal volume of PAKpili (345 g/ml), PAK 128-144 red (10 nmol/ml), PAK 128-144 ox (50nmol/ml) or PBS. The mixture was incubated at 37° C. for 1 h andagitated at 300 rpm. TECs were then collected by centrifugation (6,000×gfor 1 min at room temperature) and washed 3 times with PBS. Anti-pilusmonoclonal antibodies PK3B (0.1 ml of a 10⁻⁴ dilution of purified IgGwith a titre of 10⁸ in PBS) or monoclonal antibody PK99H (0.1 ml of a10⁻³ dilution of Purified IgG with a titre of 10⁶ in PBS) were added toTECs incubated with pili or synthetic peptides PAK 128-144 red and PAK128-144 ox, respectively (PK3B reacts with PAK pili without affectingpilus binding activity, but PK3B does not react with either PAK 128-144red or PAK 128-144 ox). Control preparations included monoclonalantibodies PK3B and PK99H incubated TECs in PBS without the presence ofpili and synthetic peptides. TECs were then collected by centrifugationand washed 3 times with PBS. Rabbit anti-mouse IgG, IgM (H+L) affinitypurified IgG conjugated to fluorescein isothiocyanate (Cedarlanelaboratories) in PBS (0.1 ml of a 1/100 dilution) was added to thewashed TECs preparations and incubated for 30 min at 37° C. and agitatedat 300 rpm. The TECs were washed 3 times as described above andresuspended in 0.1 ml of PBS. Wet mounts were prepared, and examined byepifluorescence and phase contrast microscopy using a Lietz Laborluxequipped with a MPS4 camera system. Photographs were recorded with KodakT-Max film. Results are shown in FIG. 4 wherein indirectimmunoflourescent localization of PAK pili binding to fractionatedciliated TECs A) phase contrast micrograph of TEC with bound PAK pili;B) immunoflourescent micrograph of PAK pili bound primarily to the ciliaand luminal portion of the cytoplasmic membrane of the same ciliatedTECs visualized by phase contrast microscopy in A, C and D are the phasecontrast image and the immunoflourescent image of a control TEC exposedto monoclonal antibody PK3B and FITC conjugated anti-mouse but notexposed to PAK pili. Further results are shown in FIG. 5, whereinindirect immunoflourescent localization of binding of synthetic peptidesAc17ox (A and B) and Ac17red (C and d) to human ciliated TECs.Micrographs E and F are control preparations not exposed to thesynthetic peptides but exposed to monoclonal antibody PK99H and FITCconjugated anti-mouse IgG. Figures A, C and E are phase contrastmicrographs of the same cells visualized by immunoflourescencemicroscopy in Figures B, D and F. Note that synthetic peptide Ac17ox andAc17red both bind primarily to the cilia and the luminal portion of thecytoplasmic membrane of TECs. The apparent limited binding of Ac17ox tothe TECs is due to the lesser affinity of monoclonal antibody PK99H foroxidized form of the peptide relative to the reduced form of thepeptide.

EXAMPLE 12 Pak 128-144 Binding to Bec Blots

The discontinuous sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) described by Laemmli and Favre, J. Mol. Biol.(1973) 80: 575-599 was employed. SDS-PAGE of BECs was performed asdescribed above using 8% acrylamide gels. BECs (2×10⁵ cells/ml) weresolubilized at 100° C. for 15 min in 2% (wt/vol.) SDS, 5% (vol/vol)β-mercaptoethanol, 10% (vol/vol) glycerol in 0,625 mM Tris buffer pH6.8. Solubilized BECs (25 μl) were loaded on the gel and electrophoresedat 20 mA/gel (constant current). Electrophoretically separated materialwas transferred to nitrocellulose (Schleicher & Schuell) byelectrophoretic transfer as described by Towbin et al., P.N.A.S.(U.S.A.) (1979) 76: 4350-4354. After transfer nitrocellulose blots wereblocked with 3% (wt/vol) BSA, 0.25% (wt/vol) gelatin, 0.1% (vol/vol)normal rabbit serum, 0.05% (vol/vol) Nonidet P-40, 5 mM EDTA, 150 mMsodium chloride in 50 mM Tris buffer pH 7.5 at 37° C. for at least 3 h.

Before use blots were rinsed with PBS. Blots were then incubated withPAK 128-144red (0 to 20 nmol/ml) at 37° C. shaking at 100 rpm. After 2h, blots were washed 3 times with BBBB (TTBS) (10 min per wash). Murinemonoclonal antibody PK99H (10⁻⁴ dilution in TTBS) (this monoclonalantibody recognizes the PAK 128-144 synthetic peptide) was incubatedwith the blot at 37° C. for 1 h at 100 rpm. The blot was then washed 3times with TTBS. A goat anti-mouse IgG (H+L) immunoglobin-alkalinephosphatase conjugate (Jackson Laboratories) in TTBS was added andincubated for 1 h as above. The blot was washed 3 times with TTBS andonce with Tris buffered saline. A substrate solution (NBT/BCIP)consisting of 0.33 mg/ml nitro blue tetrazolium chloride, 0.165 mg/ml5-bromo-4-chloro-3-indolyl-phosphate, 100 mM sodium chloride, 5 mMmagnesium chloride in 100 mM Tris buffer pH 9.5 was added and colordevelopment stopped by rinsing the blot in distilled water. Results areshown in FIG. 4, wherein binding of PAK pili and synthetic peptideAc17red to blotted BEC proteins on nitrocellulose is plotted. Binding ofsynthetic peptide to the immobolized proteins was assessed (followingblocking of the nitrocellulose with BSA) utilizing monoclonal antibodyPK99H (or monoclonal antibody PK3B for PAK pili) followed by standardimmunoblotting methods. PAK pili at 150 μg/ml (lane 1), Ac17red at 20(lane 2), 10 (lane 3), 5 (lane 4), or 0 (lane 5) nmoles/ml was incubatedwith the blotted BEC protein. BEC proteins oxidized by exposure to 30 mMperiodate and then reduced with borohydride before incubation with 20nmoles/ml of Ac17red (lane 6) or buffer (lane 7). Ac17red at 20nmoles/ml was initially reacted with 100 μg/ml of Fab fragments ofmonoclonal antibody PK99H (which binds to synthetic peptide Ac17red)(lane 8) or reacted with 100 μg/ml of Fab fragments of monoclonalantibody PK41C which does not bind to synthetic peptide Ac17red (lane9). Amido black stained BEC proteins (lane 10). Standard molecularweight markers stained with amido black (lane S).

EXAMPLE 13 Periodate Oxidation of Bec Blots

Periodate oxidation (30 mM periodate) and subsequent potassiumborohydride reduction of BEC blots was performed as described byWoodward et al., J. Immunol. Meth, 78: 143-153 (1985). One or two cyclesof oxidation-reduction were done on preblocked blots. Blots wereassessed for synthetic peptide binding as described above in Example 12.Results are also shown in FIG. 4.

EXAMPLE 14 Inhibition Of Pilus Binding to BECs

An equal volume of Fab fragments of affinity purified IgG specific forthe various synthetic peptide-BSA conjugates in PBS (0.1 ml at 0.3-0.61mg/ml) was added to purified PAK pili (0.1 ml at 100 ug/ml) andincubated at room temperature for 30 min. To this BECs (0.2 ml at2.0×105 BECs/ml) were added and the mixture incubated at 37° C., shakingat 300 rpm in a New Brunswick gyroshaker. After 2 h, BECs were collectedby centrifugation (13,000×g for 2 min) and washed 5 times with PBS.Monoclonal antibody PK3B (0.2 ml of a 10-4 dilution in PBS) was added tothe BEC pellet and incubated as described above for 1 h. The BECs werethen collected by centrifugation and washed 5 times with PBS. Goatanti-mouse IgG Fc-specific immunoglobin G-peroxidase conjugate (JacksonLaboratories) was added to the mixture incubated as described above for30 min. The BECs were collected by centrifugation and washed 5 timeswith PBS. The pellet was resuspended in 0.2 ml of ABTS(2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)) in citrate bufferpH 4.2+0.03% peroxide and transferred to a clean tube. The reaction wasstopped by the addition of 0.2 ml of 4 mM NaN3 and the optical densityat 405 nm was determined after removal of the BECs by centrifugation.The BEC concentration in each tube was determined with a hemocytometerat the end of the assay prior to the removal of BECs by centrifugation.

                  TABLE 5                                                         ______________________________________                                        Results of Inhibition Studies:                                                FAB Fragments Produced from Polyclonal                                        Antisera Raised in Rabbits to the                                             Synthetic Peptide 128-144 from PAK Pilin Sequence                             Protein Conc   % of             Abs                                           (mg/ml).sup.2  Control  S.D..sup.3                                                                            405 nm.sup.1                                                                         S.D.                                   ______________________________________                                        128-144 rl                                                                            0.43       72.03    4.89  0.547  0.034                                128-144 r2                                                                            0.55       62.30    0.13  0.473  0.001                                128-144 ol                                                                            0.45       70.62    2.53  0.563  0.018                                128-144 o2                                                                            0.61       35.68    7.60  0.271  0.053                                Preimmune                                                                             0.30       100.00   13.91 0.759  0.098                                PK99H   0.05       64.62    2.47  0.490  0.017                                Control 0.00       92.66    11.33 0.703  0.080                                ______________________________________                                         End point titration by ELISA of all Fabs at all concentrations shows was      approximately 10.sup.3                                                        .sup.1 Corrected for background.                                              .sup.2 Protein measured by FolinLowry using BSA as standard.                  .sup.3 Experiments were done in triplicate and whole experiments were         replicated 3 times.                                                      

EXAMPLE 15 Preparation of Fab Fragments

Fab fragments were prepared using immobilized papain (Pierce). Briefly,affinity purified antibody was dialyzed against 20 mM cysteine HCl, 10mM tetrasodium ethylenediaminetetraacetic acid (EDTA) in 20 mM sodiumphosphate buffer pH 6.2. Antibody (1 ml containing approximately 2 mgantibody) was added to 0.5

ml immobilized papain and incubated at 37° C. for 20 h with shaking at150 rpm. The immobilized papain was removed by centrifugation and thesupernatant containing the Fab fragments diluted with 1 ml of PBS. TheFab fragments were purified by HPLC using a protein G column eluted withPBS. Fab fragments were collected in the flow through, while Fcfragments were eluted from the column with 10 mM glycine pH 2.75. Fabfragments were concentrated by placing the Fab effluent in dialysis 0tubing (molecular weight cutoff of <8000) and extracting liquid from thedialysis sack using polyethylene glycol (molecular weight of15,000-20,000). The fragments were then dialyzed against PBS. Activityof Fab fragments was checked by ELISA and production of FAB fragmentsconfirmed by SDS-PAGE.

EXAMPLE 16 Effect of Fab Fragments to the Synthetic Peptide ConjugatesCorresponding to Amino Acid Sequences of Pak Pilin on Pak Pili Bindingto Human Buccal Cells (BECs)

Fab fragments were preincubated with pili before the addition of BECs(1×105 cells/mL final concentration) and pili binding was detected usingmonoclonal antibody PK3B. All Fabs were diluted such that their finaltitre as measured by ELISA to PAK pili was 10³. The results are shown inthe bar graph of FIG. 7. the bar graph demonstrates that Fab fragmentsproduced against regions other than the C-terminal of PAK pilin areineffective at preventing pilin binding to BECs. The most effectivefragments are r1, r2, 01 and 02, directed at residues 128-144, whichreduce pilin binding to 40% to 70% of the control and preimmune serum.This is similar to the effect shown by Fab 99H which is made fromanti-PAK pilin monoclonal antibody PK99H which is also directed at thisC-terminal region. This graph helps to demonstrate that the C-terminalloop region, residues 128-144, are specifically involved in

pilin binding to BECs. The legend applicable to FIG. 7 is as follows:22=Feb fragments produced against residues 22-33,41=Fab fragmentsproduced against residues 41-49, 58=Feb fragments produced againstresidues 58-70, 75=Fab fragments produced against residues 75-84, 89=Fabfragments produced against residues 89-99, 107=Fab fragments producedagainst residues 107-116, 117=Fab fragments produced against residues117-125, r1=Fab fragments produced against 0 residues 128-144 withcysteine residues in the reduced state, r2=Fab fragments producedagainst residues 128-144 with cysteine residues in the reduced states,o1=Fab fragments produced against residues 128-144 with cysteineresidues in the oxidized state, o2=Fab fragments produced againstresidues 128-144 with cysteine residues in the oxidized state, Pre=Fabfragments produced from the preimmune sera, 99H=Fab fragments producedfrom anti-PAK pilin monoclonal antibody PK99H, Cont=No Fab fragmentsadded.

Although preferred embodiments of the invention are described herein indetail, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims.

We claim:
 1. A peptide vaccine composition composed of(A) a pilinpeptide consisting of amino acid residues 128-144 of a Pseudomonas pilinprotein, including one of the sequences:(i) C T S D Q D E Q F I P K G C,(ii) C K S T Q D P M F T P K G C, (iii) C T S T Q E E M F T P K G C,(iv) C T S N A D N K Y L P K T C, or (v) C A T T V D A K F R P N G C;where the two cysteine residues (C) in the peptide are cross-linked; (B)an immunogenic carrier to which the peptide is attached; and (C) apharmaceutically acceptable diluent.
 2. The vaccine of claim 1, whereinsaid peptide consists of amino acid residues 128-144 of PAK pilinprotein and includes the sequence C T S D Q D E Q D E Q F I P K G C.